World Journal

of Microbiotogy

and Biotechnology

9, 196-201

Influence of culture density, pH, organic acids and divalent cations on the removal of nutrients and metals by immobilized Anabaena doliolum and Chlorella vulgaris N. Mallick and L.C. Rai* The potential of alginate-immobilized Anabaena doliolum and Cblorella vulgaris was assessed for removal of nutrients (NO; and NH:) and metals (Cr,O:and Ni’+) at different biomass concentrations (0.05, 0.1, 0.25, 0.49 and 1.22 g dry wt I-‘) and pH values (4 to 10). Though uptake of all these substances was higher in concentrated algal beads (0.25, 0.49 and 1.22 g dry wt I-‘), their rate of uptake was significantly (P < 0.001) lower than that of low (0.05 g dry wt I-‘) cell density beads. For A. doliolum, there was no significant difference in uptake rates for beads having densities of 0.05 and 0.1 g dry wt 1-l. Chl or ell a vulgaris, however, showed maximum efficiency at 0.1 g dry wt 1-r. Uptake of both the nutrients and the metals was maximal at pH 7 followed by pH 8, 6, 9, 10, 5 and 4. Of the different substances (organic acids and divalent cations) used, humic acid was most efficient in decreasing metal uptake. Mg2+ was, however, more efficient than Ca2+ in decreasing Ni2+ uptake. Immobilized algae with a cell density of 0.1 g dry wt 1-l were the most efficient for nutrient and metal removal at pH 6 to 8. IGY words: Anabaena dolioh,

calcium, Chlorella vu~aris, heavy metals, immobilization,

The major environmental health problems confronting both developed and developing countries are related to lack of clean water supply and proper facilities for disposal of sewage. Many aquatic ecosystems are being progressively contaminated by indiscriminate discharge of toxic metals and nutrients from industries and agriculture (Stokes 1983). Sustenance of life in such contaminated water bodies requires proper management and restoration by way of nutrient and metal removal. Of the various strategies used to achieve this end, immobilized cell technology has gained prominence in removing metals and nutrients (N, P) from contaminated environments (Chevalier & De la Notie 1985; Darnall ef al. 1986; Singh et al. 1989; Costa & Gomez 1991). This technology not only prevents the wash-out of biomass in continuous flow reactors, but offers a greater degree of operational flexibility and easy separation (Lakhawala et al. 1989). Immobilized algae are not only resistant to high concentrations of toxic metals but possess

N. Mallick and L.C. Rai are with the Laboratory of Algal Biology. Centre of Advanced Study in Botany, Banaras Hmdu U&r&, Varan&i 221005, India: fax: (91) 542 311 693. * Corresponding author. @ 1993 Rapid

196

Communications

of Oxford

Ltd

World Journal of Micmbiology and Biotechnology, Vol 9. 1993

magnesium,

organic

acids, pH.

high chlorophyll content (Robinson et al. 1985) and greater efficiency than free cells in removing metals and nutrients over repeated cycles (Bozeman ef al. 1989; Rai & Mallick 1992). Compared with other immobilizing materials, calcium alginate is becoming more attractive because the technique required is simple and low cost (Bajpai et al. 1985). The performance of such biological reactors may be affected by a number of variables, one of which is the bead design. It is well known that an algal culture cannot maintain its optimal viability for long due to shading effects if the density of the culture is very high (Abeliovich 1980). Optimal utilization of light usually requires low algal densities (De la Noiie 1983). Although most microbial assays are conducted in media at pH 7, the pH of natural water spans a wide spectrum. In biological systems, there is usually a range within which pH changes have little measurable effect, despite the fact that every organism has a specific pH requirement (Babich & Stotzky 1986). Since immobilized-cell reactors are mainly composed of biological organisms, it is presumed that functioning of such reactors would be affected by a change in pH of the medium. In addition to this, the presence of

Removal different cations and organic substances may also affect the performance of bioreactors designed for restoration of environmental quality. Hence, the objectives of this study were to find out the most effective cell density and pH for maximal operational performance of bioreactors consisting of a cyanobacterium, Anabaena doliolum and a green alga, Chlorella vulgaris. It was further intended to understand the interactive effects of naturally-occurring organic acids and divalent cations on the removal efficiency of the bioreactors.

Materials

and Methods

Anabaena daliolum and Chlorella vu&is were raised axenically, respectively, in the medium of Allen & Amon (1955) at pH 7.5 and the medium Chu-10 of Gerloff et al. (1950) at pH 6.8. Incubation was at 24 f 2°C under 72 pmol photon mm2 S-I light intensity with a photoperiod of 14 :I0 h. Stock solutions of NiCl,.6H,O and K,Cr,O, were filter-sterilized by passing through membrane filters (0.45 pm) before adding to the culture medium. Non-inhibitory concentrations (2 pg ml-‘) of organic acids (citric, humic and oxalic) and cations (Cal+ and Mg’+) were used for this

study. Immobilization of Algal Cells Algal cells were suspended in 5% (w/v) solution of sodium alginate prepared in the growth medium. Different algal cell densities were maintained

by measuring

their

absorption

at 678 nm and converted

to dry wt using the equation of Lavoie & De la Notie (1983). The sodium alginate/algal mixture was then added dropwise, from a syringe, into 0.2 M CaCl,. The alginate beads (diameter: 3.0 f 0.15 mm) formed by cross-linking with Ca’+ were harvested and washed with culture medium and resuspended in fresh growth medium. Removal of nutrients and metals at different biomass concentrations

was

estimated

withdrawn from the experimental

after

24 h.

Beads

were

also

flasks after 24 h and resuspended

in fresh medium containing nutrients/metaIs. Removal efficiency was tested at selected pH medium with MES or borax/boric acid (5 nM).

values

adjusting

Uptake of NO;, NH2 and Metals The monolayer of beads spread over the bottom of the flask was incubated in the culture room with light intensity of 72 pmol and NH: were estimated photon rn-’ s-l at 24 _+ 2°C. NO; calorimetrically by the brucine/sulphuric acid method (Nicholas & Nason 1957) and Nessler’s reagent (Herbert et al. 1971) respectively. Ni and Cr were measured by the method of Martin (1979) with an atomic absorption spectrophotometer. All the experiments were performed in triplicate in the growth medium. Uptake of nutrients and metals by immobilized algae was defined as the value obtained after subtracting the amount of metals or nutrients taken up by alginate beads (without algae) from the amount taken up by the alginate-immobilized algae.

Estimation of Carbon Fixation and O,-evolution Carbon fixation was estimated by measuring the depletion of %O, (NaH14C0,; sp. act. 6.4 x 10” Bq/mol) from the medium as described by Rai & Raizada (1986). Gross photosynthesis (02 evolution + consumption in dark) was measured with a polarographic oxygen electrode enclosed in a lo-ml reaction vessel and connected to an oxygen analyzer (Digital Oxygen System, Model 10; Rank Brothers, Cambridge, UK).

of

nutrients and metals by immobilized algae

Statistical Analysis The results were analysed and Student’s t-tests.

using

analysis

of variance

(ANOVA)

Results Upfake of NO;, NH:, Ni’+ and Cr&by Immobilized Algae Data presented in Figure I demonstrate a density-dependent removal of NO; and NH: by the immobilized algae. However, the rate of uptake was greater in low cell density beads (0.05 and 0.1 g dry wt 1-r) than those with high cell density (0.25, 0.49 and 1.22 g dry wt 1-r; Table I). Compared with the 0.05 g dry wt 1-l of A. doliolum, there was a 27%, 42% and 65% decrease in the dichromate uptake rate at cell density of 0.25, 0.49 and 1.22 g dry wt l-r, respectively. For NiZf uptake, the corresponding decreases were, however, IS%, 45% and 62%, respectively (Table I). Chlorella vtrlgaris also exhibited a similar pattern with corresponding decreases of 32%, 42% and 71% for dichromate and 42%, 54% and 69% for Ni” + uptake, respectively. Although the amount of metal taken up by 0.1 g dry wt 1-r of A. doliolum was almost double that by 0.05 g dry wt l-r, there was no significant difference in uptake rates between these two densities of beads. However, C. vulgaris showed better efficiency at a culture density of 0.1 g dry wt 1-l (Table 1). It is also clear from Table 1 that the rate of Ni” uptake was generally higher than that of dichromate. With A. doliolum, Ni’+ uptake was approximately JO%, 34%, 47%, 26% and 44% higher than Cr,Otfor cell densities of 0.05, 0.1, 0.25, 0.49 and 1.22 g dry wt i-r, respectively. For C. vulgaris, the corresponding values were, respectively, 55%, 53%, 33%. 22% and 66% higher. Chlorella vulgaris had a faster initial uptake of nutrients than A. doliolum (Figure 1). A similar pattern was also noticed for metal uptake (data not shown). While exploring the possibility of repeated use of immobilized algae, a significant decrease (F significant at P < 0.001) in removal efficiency from first to second and second to third cycles was noticed (Table 1). The reduction was, however, more pronounced for beads of low culture density (0.05 and 0.1 g dry wt 1-l) than the more concentrated ones. Eflect of Culture Density on Photosynthesis Figure 2 represents the rate of carbon fixation and 0,.evolution by immobilized algae at different culture densities. Compared with 0.05 g dry wt l-r, an increase in the rate of both processes was noticed at a cell density of 0.1 g dry wt 1-l. At higher cell densities (0.25, 0.49 and 1.22 g dry wt I-‘), however, the rate of O,-evolution of A. doliolum was reduced, respectively, by 13%, 42% and 54%. The corresponding reductions in carbon fixation were

World Jmrnal

of Microbiology

and Biotechnology. Vol 9, 1993

197

N. Mallick

and L.C. Rai

B

A. doliolum

2.5

0

4

8

12

Flgure (A)

Table cycles

1. Uptake and 0.05

of NH:

(0)

and

NO;

time

by algal

24

6

4

0

(h) beads

12 Incubation

of different

cell

densities,

i.e. with

dry

weights of

16 time

20

24

(h)

1.22 (0).

0.49

(A),

0.25 (a),

0.10

g I-‘.

1. Uptake rate 1, 2 and 3.’

of NO;,

NH:,

Cr@-

(pg NO;

NO; uptake mg dry wt-’

1

2

3

0.05

3.83

2.64

0.10

(-) 3.78

(31) 2.59

0.25

2.93

0.49

(24) 2.01

1.51

1.22

(48) 0.93 (76)

Cell density (g dry WI I-‘)

20

16

Incubation

and

h-‘)

Ni ‘+ by different

(pg NH:

NH: uptake mg dry wt-’

biomass

h-‘)

concentrations

of immobilized

Cr,O:uptake (pg Cr mg dry vv-’ h-’

1

2

3

1

1.95

10.5

7.1

4.8

0.26

(49) 2.04

t-1 10.6

(32) 7.2

(54) 5.4

(4 0.26

(32) 1.53

(47) 1.08

t-11 7.4

(31) 4.2

(49) 2.9

(60)

(72) 0.93

(30) 5.3

(60) 3.1

0.58

(76) 0.42

(50) 2.1

(70) 1.9

(85)

(89)

(80)

x 10-l)

A. do/lo/urn

and C. vulgar/s

Ni2+ uptake (pg NI mg dry wt-’ h-’

in

x 10-l)

2

3

1

2

3

0.16

0.06

0.34

0.18

0.14

(38) 0.13

(77) 0.07

(4 0.35

(48) 0.20

(59) 0.13

(0) 0.19

(50) 0.09

(73) 0.04

(-4 0.28

(41) 0.14

0.10

(72) 2.0

(27) 0.15

(65) 0.08

(85) 0.04

(18) 0.19

(59) 0.12

(71) 0.08

(61)

(42) 0.09

(69) 0.04

(85) 0.02

(45) 0.13

P-55)

1.3

0.07

(74) 0.07

w

P-3)

(65)

(85)

(92)

(62)

(79)

(79)

A. doliolum

(2)

(60)

(62)

C. vulgaris 0.05

3.01

2.45

2.01

9.10

7.21

5.01

0.31

0.25

0.19

0.48

0.39

0.26

(1% 2.39

(33) 2.03

c-1 9.32

(21)

0.10

(-1 3.19

6.98

(45) 5.22

(3 0.34

(19) 0.24

(39) 0.15

t-1 0.52

(19) 0.39

(46) 0.28

f-53)

(27)

0.25

2.63

1.95

(33) 1.75

(F-11) 8.63

(23) 6.21

(43) 4.03

t-91 0.21

(23) 0.14

(52) 0.13

(k-8) 0.28

(19) 0.21

(42) 0.18

0.49

(13) 1.95

(35) 1.13

(42) 0.81

(5) 7.45

(32) 4.03

(56) 4.01

(32) 0.18

(55) 0.13

(58) 0.11

(42) 0.22

(56) 0.15

0.14

(35) 0.81

(63) 0.62

(73) 0.44

(18)

1.22

5.03

(56) 2.10

(56) 2.00

(42) 0.09

(58) 0.06

(65) 0.06

WI 0.15

(69) 0.11

(71) 0.10

(73)

(79)

(85)

(45)

(77)

(78)

(71)

(81)

(81)

(69)

(77)

(79)

between

0.02

in parentheses

denote

% reduction

* Standard

errors

stimulation

over

198

ranged control.

F (ANOVA)

and

values

0.07. significant

Data

at p < 0.005.

World Journal of Microbiology and Bmfechnology, Vol 9, ,993

in uptake

rate;

a negative

value

(6’4

denotes

Removal

nutrients and metals by immobilized algae

lo%, 32% and 48%, respectively. a similar trend.

c. vulgaris

A. doliolum

of

Chlorella m&m-is depicted

Effect of pH on the Uptake of Nutrients and Metals Uptake of NO; and NH:, as influenced by different pH values, is summarized in Table 2. Anabaena dolioltrm had maximal uptake of both the nutrients at pH 7.0. For NO; the decrease in uptake was about 2%, 22%, 36%, 39%, 55% and 60% for pH values of 8, 9, 10, 6, 5 and 4, respectively. decreases were, For NH: uptake, the corresponding however, 2%, 19%, 29%, 40%, 55% and 58%, respectively. In contrast, C. mdgaris gave maximal removal efficiency in the range of pH 6 to 7. However, both the organisms had maximal efficiency for removal of Cr and Ni at pH 7. At all the pH values tested, the amount of NiZf absorbed was much higher than dichromate.

Figure

2. Rate

by beads 0.49 (H),

Table

of photosynthetic

O,-evolution

and carbon

of different biomass, i.e. with dry weights 0.25 (m), 0.10 (m) and 0.05 (0) g I-‘.

2. Influence

PH (pg NO;

of pH on the uptake NO; uptake mg dry wt-’

h-‘)

fixation

of 1.22 (m),

of nutrients

pg NH:

Interaction of Organic Acids (Hwnic, Citric and Oxalic) and Divalenf Cations (Ca’+ and Mg’+) with Metal Uptake Impact of organic acids and divalent cations on regulation of metal uptake is given in Table 3. In the case of immobilized A. dolioltlm, Ni uptake was decreased by 67%,

and metals

NH: uptake mg dry wt-’

by immobilized

C. vu/garis

and A. do//o/urn

Cr,O:uptake @g Cr mg dry wt-’ h-’

h-‘)

x lo-‘)

after

24 h of treatment.*

Ni*+ uptake @g Ni mg dry wt-’ h-’

A. doliolum

C. vulgaris

A. doliolum

C. vulgaris

A. doliolum

4.0 5.0

1.28 f 0.05 1.45 * 0.02

1.01 * 0.04 1.28 k 0.03

3.75 4.02

* 0.03 + 0.02

4.38 + 0.04 5.91 ) 0.03

0.07 0.14

+ 0.003 + 0.005

0.04 * 0.004 0.10 + 0.004

0.31 + 0.004 0.44 * 0.003

0.32 + 0.003 0.39 * 0.004

6.0

1.96 k 0.04

2.93 f

0.02

5.35

* 0.02

7.93 + 0.03

0.18

f 0.002

0.15

f 0.003

0.52 + 0.002

0.43 * 0.005

7.0 8.0 9.0

3.21 k 0.03 3.14 * 0.03 2.50 k 0.02

3.02 k 0.04 2.81 k 0.03 2.08 k 0.04

8.92 8.78 7.23

+ 0.03 7t 0.04 f 0.02

8.08 f 0.05 7.82 f 0.03 6.43 + 0.02

0.23 0.21 0.10

k 0.004 * 0.004 * 0.002

0.20 f 0.003 0.19 * 0.002 0.07 5 0.004

0.61 + 0.005 0.54 * 0.002 0.38 + 0.004

0.53 * 0.003 0.47 * 0.005 0.39 * 0.002

10.0

2.05 k 0.05

1.92 f 0.03

6.33

f

6.11 k 0.04

0.09 * 0.003

0.05 f 0.004

0.38 + 0.004

0.35 f

l

Values

are

means

+ SD.

Table algae

‘t’ (students’

0.03

t-tests)

significant

3. interaction of organic after 24 h of treatment.’

Combination

acids

Control C + oxalic C C C c

+ + + +

and divalent

cations

C. vulgaris

+ 0.002 * 0.003

0.023 0.016

5 0.002 + 0.001

citric acid humic acid Ca2+ k4g2+

0.013 0.011 0.021 0.021

+ + * *

0.016 0.012 0.016 0.015

f * f +

are

means

k SD. ‘t’ (students’

t-tests)

Cr,O:-

and

Ni’+

uptake

Ni*+ uptake (pg Ni mg dry wt-’

0.028 0.015

l Values C+ontrol.

with

h-‘)

acid

0.001 0.002 0.003 0.001

A. doliolum

C. vulgar/s

0.003

at p < 0.05

Cr,O:uptake (pg Cr mg dry wt-’ A. doliolum

C. vulgaris

x 10-l)

0.003 0.003 0.001 0.004

significant

A. doliolum

by test

h-‘) C. vulgaris

0.039 0.021 0.019

* 0.002 * 0.001 + 0.002

0.029 0.015 0.013

+ 0.002 &- 0.001 ) 0.002

0.013 0.031 0.027

* 0.001 f 0.001 + 0.002

0.011 0.019 0.015

+ 0.003 f 0.002 * 0.002

at p < 0.05.

World ]ournal of Microbiology and Biotechnology, Vol 9, 1993

199

N. Mallick

and

L.C. Rai

51% and 46%, and Cr,O;- uptake decreased by 61%, 54% and 66%, by humic, citric and oxalic acids, respectively. Similarly, humic acid was more efficient in decreasing the uptake of metals by C. vulgaris. Of the divalent cations, was more e&cient (10% for A. doliolum and 15% for MgC. vulgaris) than Ca’+ in decreasing the uptake of Ni. However, no significant difference was noticed between Ca’ + and Mg ‘+ in decreasing dichromate uptake.

Discussion Results presented in Table I showed a slow uptake rate for nutrients (NO; and NH:) and metals (dichromate and Ni’+) in high cell density beads (0.25, 0.49 and 1.22 g dry wt 1-l). This decrease in removal efficiency of concentrated algal beads may be due to self-shading, i.e. algae enclosed in the centre of the beads are unable to get sufficient light and, therefore, cannot perform optimal metabolic activity. A decrease in the rate of 0, evolution (Figure 2) in high density beads further indicates that light limitation may be considerable and lead to ATP depletion which could then affect nutrient uptake (Oelmuller et al. 1988; Rai et al. 1992). A very fast initial uptake of nutrients and metals, as noticed from the present results, may be due to availability of free binding sites. A fast initial uptake by C. vulgaris may be attributed to its unicellular nature, which facilitates absorption by the entire cell surface. However, a gradual fall in uptake rate from first to subsequent cycles emphasized the significance of using fresh algal beads for nutrient and metal removal. Table 2 demonstrates a substantial decrease in metal and nutrient removal efficiency by these biological reactors, both at acidic and alkaline pH compared with that at pH 7. A decrease in uptake of metals at high pH due to their precipitation as metal hydroxides (Borgmann 1983), on the one hand, and competition between metal and hydrogen ions for binding at low pH (Ali & Deo 1991), on the other, cannot be ruled out. Of various organic acids used, humic acid was the most effective in reducing metal uptake (Table 3), apparently because of its multiple binding sites for metal cations (Hongve et al. 1980). High concentrations of Ca’+ and Mg’+ are characteristic features of hard and eutrophic waters. Decreased metal uptake in the presence of Ca2+ and MgZf (Table 3) is a consequence of competitive inhibition (Schecher & Driscoll 1985). Mg’+ was, however, found more efficient in decreasing the uptake of Ni’+. This could be due to the similarity in the chemistry and ionic radius of Mg’+ and Ni’+; Ni’+ is known to bind in place of Mg’+ (Hughes & Poole 1989). This study recommends the use of low cell density (0.1 g dry wt 1-l) beads and a pH range of 6 to 8 for maximal operational performance of immobilized algae. However, before using such algae for metal and nutrient removal, the

200

World ]oumal of Microbiology

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presence of organic acids and divalent cations (which may impede the removal efficiency because of their greater affinity for metals) should be ascertained.

Acknowledgement The financial assistance from The University Grants Commission, New Delhi, in the form of a Career Award to LCR and a Research Associateship to NM, is thankfully acknowledged.

References Abeliovich, A. 1980 Factors limiting algal growth in high rate oxidation ponds. In Algal Biomass, eds Shelef, G. & Soeder, C.J. pp. 205-372. Amsterdam: Elsevier/North Holland Biomedical. Ali, M. & Deo, N. 1991 Effect of pH on adsorption process of chromium (vi) with a new low cost adsorbant. Indian ]otrrnal of Environmental Protection 12, 202-209. Allen, M.B. & Amon, D.I. 1955 Studies on nitrogen-fixing blue green algae. I. Growth and nitrogen fixation by Anabaena cylindrica

Lemm.

Plant Physiology

30. 366-372.

Babich, H. & Stotzky, G. 1986 Environmental factors affecting the utility of microbial assays for the toxicity and mutagenicity of chemical pollutants. In Toxicity Testing wing Microorganisms, eds Dutka, B.J. & Bitton, G. pp. 9-42. Boca Raton, FL: CRC Press. Bajpai, P.K., Wallace, J.B. & Margaritis, A. 1985 Effect of calcium chloride concentration on ethanol production and growth of immobilized Zymomonas mobilis. Journal of Fermentation Technology

63,

199-203.

Borgmann, U. 1983 Metal speciation and toxicity of free ions to aquatic biota. In Aqtratic Toxicology, ed Nriagu, J.O. pp. 47-72. New York: John Wiley and Sons. Bozeman, J., Koopman, B. & Bitton, G. 1989 Toxicity testing using immobilized algae. Aquatic Toxicology 14, 345-352. Chevalier, P. & De la Notie, J. 1985 Wastewater nutrient removal with microalgae immobilized in carrageenan. Enzyme and Microbial Technology 7, 621-624.

Costa, A.C.da & Gomez, S.G.F. 1991 Metal biosorption by sodium alginate immobilized Chlorella homosphaera cells. Biofechnology Lefters 13, 559-562. Damall, D.W., Greene, B., Benzl, M.T., Hosea, J.M., McPherson, R.A., Snedden, J. & Alexander, M.D. 1986 Selective recovery of gold and other metal ions from an algal biomass. Environmental Science and Technology 20, 206~208. De 1aNoiie, J. 1983 Hyperintensive wastewater tertiary treatment by floculated activated algal sludge. In Proceedings oflnternationa! Conference on the Commercial Applications and lmplicafions of Biofechnology, London, UK, pp. 1005-1015. London. Gerloff, G.C., Fitzgerald, G.P. & Skoog, F. 1950 The isolation, purification and culture of blue-green algae. American Journal of Botany 37, 216-218. Herbert, D., Phipps, P.J. & Strange, R.E. 1971 Chemical analysis of microbial cells. In Methods in Microbiology, Vol. 5B, eds Norris, J.R. & Ribbons, D.W. pp. 205r344. London: Academic Press, Hongve, D., Skogheim, O.K., Hindar, A. & Abrahamsen, H. 1980 Effects of heavy metals in combination with NTA, humic acid and suspended sediment on natural phytoplankton photosynthesis. Bulletin of Environmental Contamination and Toxicology 25,

594-600.

Removal Hughes, M.N. & Poole, R.K. 1989 Metal mimicry and metal limitation in studies of metal-microbe interactions. In Metal-Microbe Interactions, eds Poole, R.K. & Gadd, G.M. pp. l-18. New York: IRL Press. Lakhawala, F.S., Lodaya, M.P. & Yang, C. 1989 Design of toxic waste treatment bioreactor: viability studies of microorganisms entrapped in alginate gel. In Intemationa2 Conference on Physiochemical and Biochemical Detoxification of Hazardous Wastes, Vol. 1, ed Wu, Y.C. pp. 587-599. Technomic Publishing. Lavoie, A. & De la Noiie, J. 1983 Harvesting microalgae with chitosan. loxma] of the World Marirulfure Society 14, 685-694. Levine, I.N. 1988 Transport process. In Physical Chemisfy, ed Levine, I.N. pp. 467-505. Singapore: McGraw Hill. Martin, J.H. 1979 Bioaccumulation of heavy metals by littoral and pelagic marine organisms. US Environmental Protection Agency Report No. 60013-77-038. Nicholas, DJ. & Nason, A. 1957 Determination of nitrate and nitrite. Methods in Enzymology 3, 981-984. Oelmuller, R., Schuskr, C. & Mohr, H. 1988 Physiological characterization of a astirdic signal required for nitrate and nitrite reductase. Planta 174, 75-83. Rai, L.C., Dubey, SK. & Mallick, N. 1992 Influence of chromium on some physiological variables of Anabnena doliolum. Interaction with metabolic inhibitors. Biometals 5, 13-16.

of

nutrients and metals by immobilized algae

Rai, L.C. & Mall&, N. 1992 Removal and assessment of toxicity of Cu and Fe to Anabaena dolioltlm and Chlorella uulguris using free and immobilized cells. World Journal of Microbiology and Biotechnology 8, 11~114. Rai, L.C. & Raizada, M. 1986 Nickel-induced stimulation of growth, heterocyst differentiation ?O, uptake and nitrogenase activity in Nostoc muscorum. New Phytologist 104, 111-114. Robinson, P.K., Dainty, A.L., Goulding, K.H., Simpkin, I. & Trevan, M.D. 1985 Physiology of alginate immobilized Chlorelb. Enzyme and Microbial Technology 7, 212-216. Schecher, W.D. & Driscoll, C.T. 1985 Interackion of copper and lead with Nostoc muscorum. Water, Air and Soif Pollufion 24,

85-101. Singh, S.P., Verma, SK., Singh, R.K. & Pandey, P.K. 1989 Copper uptake by free and immobilized cyanobacterium. FEMS Microbiology Letters 60, 193-196. Stokes, P.M. 1983 Responses of fresh water algae to metals. Progress in Phycological Research 2, 87-109.

(Received in revised form Sepfember 1992)

World ]ournal

of

4 September

1992; accepted 16

Microbiology and Biotechnology. Vol 9, 1993

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Influence of culture density, pH, organic acids and divalent cations on the removal of nutrients and metals by immobilized Anabaena doliolum and Chlorella vulgaris.

The potential of alginate-immobilized Anabaena doliolum and Chlorella vulgaris was assessed for removal of nutrients (NO inf3 (sup-) and NH inf4 (sup+...
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